I. Signal transduction in Platelet Activation Elevation of cytosolic calcium plays a major role in platelet activation by physiologic agonists or by extended storage. The rise in cytosolic calcium induces a variety of biochemical responses including tyrosine phosphorylation and dephosphorylation of specific proteins. Activation of platelets leads to an expression of a fibrinogen-binding integrin on the surface of platelets, followed by binding of a bivalent fibrinogen molecule and crosslinking of adjacent platelets by fibrinogen to produce a platelet aggregate. Additional tyrosine phosphorylation of internal platelet proteins takes place after aggregation and is thought to stabilize the forming aggregate. We are investigating the identity of the tyrosine phosphorylated proteins and the tyrosine kinases and phospahatses that participate in this process. II. Nitric Oxide and sickle cell disease platelets Nitric oxide (NO) mediates relaxation of arterioles and inhibits platelet activation. Excessive thrombosis found in certain clinical conditions may be due to an insufficient availability of nitric oxide. Sickle cell disease is associated with frequent pain crises the etiology of which is associated with frequent vasoocclusive phenomenon. Recently the focus of clinical investigations has turned to the role of nitric oxide in the pathophysiology of this disease. We have compared the responsiveness of platelets isolated from sickle cell patients and normal volunteers to platelet agonists and to chemical NO donors. Sickle cell platelets appear to be more responsive to NO donors which suggests that physiologic production of NO in sickle cell patients may be decreased. III. Prion Proteins and Platelets The prion protein (PrPC) is expressed on many cell types and tissues and is thought to be the infectious agent in the fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE). We have investigated the role of PrPc in platelet physiology and found that platelet PrPc appears to be associated with an intracellular membrane vesicle in resting platelets and that platelet activation increases PrPc expression on the platelet surface. The surface expression of PrPc, as detected by flow cytometry with a PrPc-specific monoclonal antibody, increased more than twofold after platelet activation. Further studies with blood cells from PrPc-knock out mice will be performed to investigate the function of PrPc in platelet physiology. We are also investigating the ability of platelets to carry infectivity from scrapie infected hamsters to healthy animals in an attempt to model the risk of transmitting TSE diseases in humans by platelet transfusion. Hamster platelets and leukocytes express none or very low quantities of monomeric PrPc either on their surface or inside the cells, as detected by a monoclonal antibody against PrPc (3F4). This is in contrast to human platelets and leukocytes, both of which carry the prion protein on their surface. To determine whether TSE infectivity is associated with platelets or leukocytes, we isolated these cells from symptomatic scrapie-infected hamsters and inoculated them into the brains of healthy hamsters. Preliminary results suggest that platelets of scrapie infected hamsters contain little, if any, infectivity while infectivity in leukocytes is enriched. Additional infectivity studies will be carried out to determine whether leukoreduction of blood can decrease the transfer of infectivity. A PrPc-knock out mice was obtained to study the role of PrPc in the transfer of infectivity. Endothelial cells express prion protein. We are utilizing a cell culture system to study whether these cells can release prion protein into medium as cellular microparticles and if this is the mechanism for releasing TSE infectivity into plasma.